Analysis of an inhibin preparation reveals apparent identity between a peptide with inhibin-like activity and a sperm-coating antigen

The amino acid sequence of a large form of inhibin-like peptide in human seminal plasma was determined, and compared with structures reported for similar inhibin preparations and a seminal plasma globulin. The data confirm and correlate previous reports on this form of inhibin-like peptide. The structural comparisons further suggest that the peptide is closely similar to or possibly identical to a sperm-coating antigen reported to be synthesized from prostatic epithelium. This may correlate with non-gonadal origins of inhibin-like material and will help to elucidate the biological roles of inhibin(s).     

Molecular weight forms of inhibin a and inhibin B in the bovine testis change with age

To investigate alterations in the molecular weight forms of inhibin in bull testis from the infantile (4-5 wk of age) to postpubertal (49-56 wk of age) periods, testicular homogenates were obtained from animals of various ages and fractionated by a combination of immunoaffinity chromatography and SDS-PAGE. Subsequently, the fractions eluted from the SDS gels were assayed for total inhibin, inhibin A, and inhibin B by fluoroimmunoassay or immunofluorometric assays (IFMAs) and for inhibin bioactivity by an in vitro bioassay. The molecular mass patterns of inhibin A and inhibin B in the testis, as determined by the dimer-specific IFMAs, showed the presence of a peak of approximate 47 kDa until 21-26 wk of age. However, the peak disappeared after 31-32 wk of age. As bulls aged, especially after 31-32 wk of age, inhibin A and inhibin B levels increased in the molecular mass region of 27-34 kDa. Total inhibin showed two peaks, of between 20 and 26 kDa and at approximately 47 kDa, until 21-26 wk of age and a single peak between 20 and 30 kDa after 31-32 wk of age. The eluted fractions corresponding to 29, 31, or 47 kDa gave a dose-response curve that was parallel to the curve generated with 32-kDa inhibin A or 29-kDa inhibin B standard in the IFMA for inhibin A or inhibin B. The fractions corresponding to 29 and 31 kDa suppressed basal release of FSH from rat pituitary cells, but the 47-kDa fraction had a lower FSH-suppressing activity. In the testes of older bulls, immunoblot analysis revealed the presence of a 29-kDa band cross-reacting with inhibin alpha and inhibin betaB antibodies and of a 31-kDa band cross-reacting with inhibin alpha and inhibin betaA antibodies. The 47-kDa band was recognized by the alpha, betaA, and betaB antibodies. Immunohistochemisty of the testis at each age showed that inhibin alpha subunits were found exclusively in Sertoli cells, but the intensity of immunostaining diminished in older bulls, in parallel with the decrease in the testicular concentrations of total inhibin. We conclude that 1) bovine Sertoli cells produce both inhibin A and inhibin B, 2) inhibin production in Sertoli cells during the prepubertal period is characterized by the 47 kDa inhibin-related material that contains precursor forms of inhibin A and inhibin B, and 3) the proportion of the mature forms of inhibin A and inhibin B increases as bulls age, although total inhibin production in Setroli cells decreases.     

Active immunization with a synthetic fragment of pig inhibin alpha-subunit increases ovulation rate and embryo production in superovulated ewes but season affects its efficiency.

Two experiments were designed to determine the effects of active immunization against one of two synthetic peptides from humans (inhibin-like peptide) or pigs (inhibin alpha-subunit) on antibody titres, ovulation rate and embryo production in ewes superovulated with 16 U ovine FSH. In Expt 1, during the breeding season, 30 ewes were subdivided into three groups: group I served as the non-immunized control; group II was immunized against inhibin-like peptide (100 micrograms inhibin-like peptide equivalent, followed by three booster injections); group III was immunized against pig inhibin alpha-subunit conjugated to human serum albumin (96 micrograms for the primary administration and 46 micrograms for the booster). In Expt 2, the efficiency of immunization against pig inhibin alpha-subunit on ovarian response and embryo production was evaluated during the non-breeding season in two groups of ewes (n = 12): group IV was a non-immunized control; Group V was immunized against pig inhibin alpha-subunit. During the breeding season, the ewes immunized against pig inhibin alpha-subunit showed higher antibody titres compared with the group immunized against inhibin-like peptide (P < 0.01) and a significant increase in ovulation rate (12.1) compared with both the control (5.0; P < 0.05) and the inhibin-like peptide-immunized group (3.1; P < 0.01). Immunization against pig inhibin alpha-subunit increased transferable embryo yield 4.5-fold (6.7 versus 1.5; P < 0.01) and improved embryo quality (94.6 versus 40.6%; P < 0.01). During the non-breeding season, immunization against pig inhibin alpha-subunit enhanced ovulation rate from 2.6 in the controls to 9.4 (P < 0.01) but did not affect transferable embryo production (3.9 versus 2.1; P > 0.05) and significantly lowered their quality (54.1 versus 100%; P < 0.01). In conclusion, active immunization against pig inhibin alpha-subunit can improve superovulatory response during the breeding season, while it appears to be unable to increase embryo yield during the seasonal anoestrus.     

Validation of coupled bioassay for inhibin by pituitary cell culture assay

Studies on the characterization of inhibin and inhibin-like factors have depended for the most part on the classicalin vitro pituitary cell culture assay. A major drawback with this assay is the turn-around time which is in the order of two weeks and consequently slows down purification efforts. The 24 h bioassay for inhibin has been found to be sufficiently sensitive and also statistically valid. Unfortunately, based as it is on a secondary response, ambiguities arise in interpreting the results. By including a parallel assay in which the mice are primed with human menopausal gonadotropin rather than human chorionic gonadotropin, it was possible to device the coupled bioassay. This enables distinguishing inhibin-like factors acting to suppress pituitary follicle stimulating hormone output from those acting at the level of gonads. In this study the coupled assay for inhibin has been compared with the classical pituitary cell culture assay in order to assess its biological and statistical validity. The data validates the bioassay on both the above counts and when considered in conjunction with the short turn-around time suggests that this assay can be highly useful in studies on isolation of inhibin from various sources.     

Identification of a heparin-binding protein in bovine seminal fluid as tissue inhibitor of metalloproteinases-2

Presence or absence of three distinct bovine seminal heparin-binding proteins (21-31 kDa) recognized in sperm extracts by a monoclonal antibody, M1, is a diagnostic indicator of fertility differences among bulls producing normal semen. We recently identified a 31 kDa fertility-associated antigenin bovine seminal fluid as a unique DNase I-like protein. We now report purification and identification of a 24 kDa seminal heparin-binding protein (HBP-24) recognized by M1. N-terminal microsequence analysis of HBP-24 purified from seminal fluid yielded 20 amino acid residues that displayed 90% identity to the N-terminus of a bovine metalloproteinase inhibitor identified as tissue inhibitor of metalloproteinases-2 (TIMP-2). A single immunoreactive band migrating at 24 kDa was detected in Western blots of cauda epididymal sperm extracts following incubation with purified seminal heparin-binding proteins and subsequent washing in vitro, indicating TIMP-2 bound to sperm membranes. Expression of TIMP-2 mRNA was detected by RT-PCR in bovine bulbourethral gland, prostate, and seminal vesicles. Mobility of the 24 kDa heparin-binding protein increased under nonreducing SDS-PAGE to approximately 21 kDa, characteristic of the reported molecular mass of TIMP-2. To our knowledge, this is the first report of TIMP-2 binding to spermatozoa and of TIMP-2 mRNA expression in bovine accessory sex glands. These results corroborate previous reports regarding the site of production of heparin-binding proteins that are related to bull fertility, and suggest that TIMP-2 influences fertility of bulls, either through inhibition of metalloprotease activity in semen or via undefined activities independent of matrix metalloproteinase (MMP) inhibition.     

Partial amino acid sequence of a human seminal plasma peptide with inhibin-like activity

An extract of human seminal plasma was found to have inhibin-like activity. The active factor was purified to homogeneity by ion exchange chromatography, molecular sieving and high performance liquid chromatography. The purified material has a mass of approximately 5 kDa and is very basic. Amino acid analysis showed the presence of approximately 35 residues while the sequencing data allowed the determination of the N-terminal 31 amino acids. There is a possibility of an additional 2–4 residues at the C-terminus, which could not be determined.     

Purification and characterization of glycolactin, a novel glycoprotein from bovine milk

A novel glycoprotein designated glycolactin, with a molecular weight of 64 kDa, a sequence hitherto unknown in the literature and capable of inhibiting the hemagglutinating activities of soybean lectin and Ricinus communis agglutinin 120, was isolated from bovine milk. Its lectin-inhibiting activity differed from that of lactoferrin, another milk protein. Like other milk proteins, glycolactin inhibited superoxide formation in vitro. Glycolactin inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of about 31 nM. It exhibited ribonucleolytic (RNase) activity towards yeast transfer RNA with a pH optimum of 7.5, and specific RNase activity towards poly C. The purification protocol of glycolactin involved removal of globulin from the acid whey fraction of bovine milk by precipitation with 1.8 M (NH4)2SO4, and adsorption on the ion exchangers CM-Sepharose and Mono S. Deglycosylation of glycolactin using glycopeptidase F produced only a slight decrease of 4 kDa in the molecular weight of glycolactin.     

Rat ventral prostate as an independent source of inhibin-like material

Castration had no effect on prostatic inhibin-like activity as compared to normal controls. Consequent upon castration there is a significant reduction in the inhibin content of the ventral prostate on a per gland basis. However, when expressed as a function of protein content, there is no change. The data suggest that the very same epithelial cells which under the influence of androgen carry out the characteristic exocrine secretory activity are responsible for the synthesis and secretion of inhibin-like material.     

Effect of immunizing prepuberal lambs of low and high ovulation rate genotypes with inhibin partially purified from bovine follicular fluid

Active immunization of prepuberal lambs with a partially purified inhibin preparation, isolated from bovine follicular fluid, increased the ovulation rate. In ewe lambs of a low fecundity breed (Suffolk x Galway), the ovulation rate rose from 1.15 to 1.95 (P<0.05) compared with that of the controls. An ovulation rate of 3.38 was recorded for immunized ewe lambs of a high fecundity breed (Finn x Dorset Horn), while the rate for mature ewes from the same flock was 2.29. Immunization did not affect the time of onset of puberty or estrous cycle length. Following immunization, antibodies were produced that bound to a pure preparation of 68kDa bovine inhibin. This report demonstrates the production of antibody to a 68kDa preparation of inhibin following active immunization of sheep using a partially purified preparation. It was concluded that the increased ovulation rate was due to the production of antibodies to inhibin, which may have reduced its negative feedback effect of FSH secretion.     

Inhibin and inhibin alpha-chain precursors are produced by immature rat Sertoli cells in culture.

Stimulation of Sertoli cells from immature rats with dibutyryl cyclic (dbc) AMP resulted in a decrease in the ratio of inhibin biological (B):immunological (I) activities in vitro. To establish the basis for this decrease, culture medium from Sertoli cells stimulated with dbcAMP was fractionated by dye-affinity chromatography, reverse-phase HPLC, and preparative PAGE. Two peaks of inhibin activity were identified: a predominantly bioactive 29-kDa peak I material (B:I ratio = 5.0) and a bio-inactive, immunoactive 27-kDa peak II material (B:I ratio = 0.1). Evidence of a subunit structure was established by iodination and immunopurification using an inhibin alpha-subunit antiserum. On reduction, peak I (29-kDa) material showed bands of 19 kDa and 14 kDa, whereas peak II (27-kDa) material showed a single 20-kDa band. On the basis of HPLC retention position, molecular mass, evidence of subunit structures and their molecular masses, and inhibin in vitro bio- and immunoactivities, peak I and II materials were most likely inhibin and the alpha-subunit precursor protein pro-alpha C. Western blotting of Sertoli cell culture medium using antiserum directed against the NH2 terminal region (alpha N) of the alpha-subunit precursor also indicated the presence of 24-kDa alpha N. It is concluded that after dbcAMP stimulation, Sertoli cells produce 29-kDa inhibin and the alpha-subunit precursor proteins pro-alpha C and alpha N. The production of the alpha-subunit precursor in addition to inhibin provides an explanation for the decrease in the inhibin B:I ratio following dbcAMP stimulation of Sertoli cells in culture.     

Isolation, characterisation and crystallization of deoxyribonuclease I from bovine and rat parotid gland and its interaction with rabbit skeletal muscle actin

A purification procedure is described yielding DNase I from bovine and rat parotid glands of high homogeneity. The apparent molecular masses of the DNases I isolated have been found by sodium dodecyl sulfate/polyacrylamide gel electrophoresis to be 34 and 32 kDa for bovine and rat parotid DNase I, respectively, and thus differ from the enzyme isolated from bovine pancreas (31 kDa). By a number of different criteria concerning their enzymic behaviour, the isolated enzymes could be clearly classified as DNases I, i.e. endonucleolytic activity preferentially on native double-stranded DNA yielding 5'-oligonucleotides, a pH optimum at about 8.0, the dependence of their enzymic activity on divalent metal ions, their inhibition by 2-nitro-5-thiocyanobenzoic acid and by skeletal muscle actin. Comparison of their primary structure by analysis of their amino acid composition and also two-dimensional fingerprints and isoelectric focusing indicate gross similarities between the enzymes isolated from bovine pancreas and parotid, but distinct species differences, i.e. between the enzymes isolated from bovine and rat parotid. All the DNases I are glycoproteins. From bovine parotid DNase I crystals suitable for X-ray structure analysis could be obtained. The DNases I from both parotid sources specifically interact with monomeric actin forming 1:1 stoichiometric complexes. Their binding constants to monomeric actin differ, being 2 X 10(8) M-1 and 5.5 X 10(6) M-1 for bovine and rat parotid DNase I, respectively. Only the enzyme isolated from bovine sources is able to depolymerize filamentous actin.     

De novo biosynthesis and localization of immunoreactive inhibin (10.7 kDa) in normal human stomach.

We have previously reported the occurrence of inhibin-like peptide in gastric juice of normal men. In the present investigation, normal gastric mucosa was shown to synthesize inhibin, in vitro, as measured by 3H-leucine incorporation (Maximum at 18 h). Furthermore, the immunohistochemical localization studies demonstrated its presence in the acid secreting parietal cells and basal region of foveolar epithelium of gastric mucosa. Surprisingly, the protein secreting zymogen cells remained unstained.     

Amino acid sequence analysis of the proteolytic cleavage products of the bovine immunodeficiency virus Gag precursor polypeptide.

Bovine immunodeficiency virus Gag proteins were purified from virions, and their amino acid sequences and molecular masses were determined. The matrix, capsid, and nucleocapsid (MA, CA, and NC, respectively) and three smaller proteins (p2L, p3, and p2) were found to have molecular masses of 14.6, 24.6, and 7.3 and 2.5, 2.7, and 1.9 kDa, respectively. The order of these six proteins in the Gag precursor, Pr53gag, is NH2-MA-p2L-CA-p3-NC-p2-COOH. In contrast to other retroviral MA proteins, the bovine immunodeficiency virus MA retains its N-terminal methionine and is not modified by fatty acids. In addition, the bovine immunodeficiency virus NC migrates as a 13-kDa protein in denaturing gel electrophoresis; however, its molecular mass was determined to be 7.3 kDa.     

The NH2-terminal residues of rat liver proteasome (multicatalytic proteinase complex) subunits, C2, C3 and C8, are N alpha-acetylated

Rat liver proteasome (multicatalytic proteinase complex) is a 20S-ring shaped particle having a molecular mass of 750 kDa, and is composed of at least 13 non-identical components ranging from 21 to 31 kDa in size. We found here that the NH2-terminal residues of all the known 13 components, except for C5, are not reactive to phenylisothiocyanate. Among them, components C2, C3 and C8 are blocked in their NH2-termini with N alpha-acetyl-Met, N alpha-acetyl-Ala, and N alpha-acetyl-Ser, respectively. The NH2-terminal portions of C2, C3, and C8 exhibit sequence similarity to one another, but that of the non-blocked component C5 differs from those of C2, C3, and C8.     

Inhibin-like and gonadotropin-like immunoreactivity in pituitary cells of male monkeys (Macaca fascicularis,Macaca mulatta)

Summary Inhibin-like immunoreactivity was detected by immunocytochemistry in the pituitaries of untreated male crab-eating macaques (cynomolgus monkey) and rhesus monkeys, in rhesus monkeys actively immunized against FSH, and in one orchidectomized crab-cating macaque. Localizations were performed by the immunogold-silver staining with 5-nm colloidal gold-conjugated second or third antibodies and by the alkaline phosphatase-anti-alkaline-phosphatase technique. Two different inhibin-specific antisera, raised against the -subunit or the entire inhibin molecule, provided identical staining patterns. Positive label was confined to the pars distalis of the pituitary and occurred exclusively in the cytoplasm of morphologically different cell types throughout the pars distalis in all pituitaries. Staining was most prominent in clusters of chromophobic cells. The presence of inhibin-like activity in the pituitary of an orchidectomized monkey with undetectable serum inhibin levels suggests that inhibin is produced within the pituitary gland. Co-localization studies for the -subunits of the gonadotropic hormones revealed that on average 82% of the gonadotropes were bihormonal. Using the same protocol, co-localization of inhibin-like activity with gonadotropin-like immunoreactivity revealed only a small degree of common distribution (<15%). Inhibinpositive cells were frequently in close proximity to gonadotropic cells and, thus, paracrine effects of inhibin on gonadotropin-synthesizing cells are conceivable.     

Monoclonal antibodies to a proenkephalin A fusion peptide synthesized in Escherichia coli recognize novel proenkephalin A precursor forms

Monoclonal antibodies have been generated to a chimeric peptide comprised of Escherichia coli beta-galactosidase fused to the amino acid sequence 69-207 of human preproenkephalin A. Two monoclonal antibodies, PE-1 and PE-2, were identified by their ability to recognize the same segment of proenkephalin A fused to the cII gene product of the E. coli bacteriophage lambda. The binding domains of PE-1 and PE-2 have been broadly located, with respect to the primary translation product, within the amino acid sequences 152-207 and 84-131, respectively. Immunoblot analysis of total bovine adrenomedullary chromaffin granule lysate reveals PE-1 and PE-2 immunoreactive forms of observed molecular mass 35, 33, 29, 24, 22, and 15 kDa, and an 18-kDa PE-1 immunoreactive form. Separation of granule membranes from their contents reveals differential membrane association of these high molecular weight polypeptides. There is preliminary evidence that PE-1 may be detecting a subset of polypeptides where shortening from the NH2 terminus has occurred. We postulate that the 35-kDa form represents the intact bovine enkephalin precursor of predicted molecular mass 27.3 kDa. This experimental approach should be generally applicable to the generation of antibodies which will recognize intact peptide precursors together with their post-translational cleavage products.     

Immunoreactive inhibin in plasma, amniotic fluid, and gonadal tissue of male and female chick embryos.

Immunoreactive inhibin was measured in plasma, amniotic fluid, gonads, and Wolffian bodies (mesonephros) of male and female chick embryos during the last week of their 21-day incubation period. The antiserum used was raised against bovine 31-kDa inhibin and was validated for RIA of inhibin in the chicken. Amniotic fluid concentrations of immunoreactive inhibin were relatively low and remained constant between Days 14 and 19. Plasma concentrations, in contrast, were high on Day 14 but declined steeply thereafter. Significantly higher plasma concentrations were noted in male than in female embryos and an even more pronounced sex difference was observed for the gonadal inhibin content. On Day 21, testes contained approximately 35 times more immunoreactive inhibin than ovaries. Surprisingly, inhibin contents in testes and male Wolffian bodies increased rather than decreased towards the end of the incubation period, indicating that gonadal and plasma inhibin concentrations are regulated, at least in part, independently. It is concluded that the chick embryo presents a convenient model for study of the secretion, the control, and the role of inhibin from fetal origin. The sex difference in plasma and gonadal inhibin suggests a differential role of inhibin in the development of the reproductive system of both sexes.     

Immunohistochemical detection of inhibin in the gonad

Antiserum to inhibin was produced in rabbits by immunization with a synthetic [Tyr30]alpha-chain(1-30)NH2 fragment of porcine inhibin coupled to bovine serum albumin, and the elicited antiserum was used in conjunction with the avidin-biotin immunoperoxidase procedure to localize inhibin-reactive cells in various rat tissue preparations. In the testes, only the Sertoli cells revealed immunoreactivity with the antiserum. Intense staining was also observed in ovarian follicular granulosa cells but not in the theca layer outside the basement membrane. In addition, the luteal cells in the corpus luteum were also stained by the antiserum. The positive staining in the gonadal tissues could be blocked completely by pre-adsorbing the serum with either the synthetic peptide or native inhibin. Immunostaining was not detected in brain, pituitary, thymus, stomach, pancreas, kidney and adrenal section, thus confirming that inhibin is a polypeptide originating only from specific cells of the gonad.     

Affinity of placental decorin for collagen

Decorin was isolated from 7 M urea extract of bovine placental cotyledons by ion-exchange and hydrophobic chromatography. Decorin and its core protein showed a broad band at about 115 kDa and a single band at 47 kDa, respectively by SDS-PAGE. Anti-decorin core protein antiserum from pig skin was reacted with placental decorin and its core protein in western blotting. The NH2-terminal amino acid sequence of core protein from placental cotyledons was not different from that of core protein from skin and bone. Glycosaminoglycan of decorin was identified as dermatan sulfate by electrophoresis on a cellulose-acetate membrane and chondroitinase digestivity. Decorin bound to collagen in the order for type III, I, and V.     

Cell-free translation and characterization of corneal keratan sulfate proteoglycan core proteins.

Bovine corneal keratan sulfate proteoglycan (KSPG) contains two core proteins, 37 and 25 kDa, if fully deglycosylated, but 47 and 35 kDa, respectively, after endo-beta-galactosidase (Funderburgh, J. L., and Conrad, G. W. (1990) J. Biol Chem. 265, 8297-8303). Chicken corneal KSPG released a single core protein of 47 kDa after endo-beta-galactosidase, and of 35 and 36 kDa, if deglycosylated with N-glycanase or trifluoromethanesulfonic acid. Affinity purified rabbit antibodies against each KSPG recognized only the intact proteoglycan or its core proteins in immunoblots of unfractionated guanidine-HCl extracts of whole cornea after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Affinity purified antibody to a synthetic peptide duplicating the NH2-terminal sequence of the 37-kDa bovine core protein showed little reactivity with untreated corneal extract but reacted with the 47-kDa bovine protein in endo-beta-galactosidase-treated extracts. RNA was isolated from bovine and chick corneal stromas and used for in vitro translation. Antibody against bovine KSPG immunoprecipitated two proteins of 56-53 kDa and a protein of 41 kDa after translation of bovine RNA. Translation of chick RNA produced a double band of 38-39 kDa and a single band of 25 kDa precipitating with antibody against chicken KSPG. Homologous unlabeled KSPG competed for binding of antibodies to these translation products. These data suggest that in vertebrate corneas, the multiple KSPG core protein isoforms may arise as products of separate mRNAs, rather than from proteolytic processing of a large polypeptide precursor.